Paraoxonase 1 arylesterase activity and mass are reduced and inversely related to C-reactive protein in patients on either standard or home nocturnal hemodialysis.

UNLABELLED: Human serum paraoxonase (PON1) activity is reduced in standard hemodialysis (SHD) (4 hours, 3 days/week) patients. Home nocturnal hemodialysis (HNHD) (8 hours, 6 days/week), provides a greater dialysis dose resulting in a greater clearance of metabolites. Whether improvements in the metabolic milieu of HNHD patients results in different PON1 activity levels compared to SHD patients is unclear. We determined serum PON1 mass and arylesterase activities in a group of HNHD patients and compared them to SHD patients and a group of healthy controls (HC). PATIENTS AND METHODS: We measured PON1 arylesterase activity and mass, C-reactive protein (CRP), cystatin C, total and high-density lipoprotein (HDL) cholesterol, triglycerides, apolipoproteins A-I and B in 15 HNHD, 15 SHD and 15 HC participants. RESULTS: PON1 arylesterase activity (p < 0.001) and mass (p < 0.05) were significantly higher in HC participants compared to SHD and HNHD participants, although no significant differences were noted between HD groups. CRP (p < 0.05) was significantly higher in SHD compared to HC participants and there were no significant differences noted between HD groups. Cystatin C (p < 0.001) was significantly different among the 3 groups. There were no significant differences noted in any lipoprotein parameters among the groups. PON1 activity (r = -0.636, p < 0.001) and mass (r = -0.425, p = 0.019) were inversely correlated with CRP in HD patients. CONCLUSION: PON1 is reduced in HNHD patients compared to HC subjects, independent of the concentration of HDL cholesterol. Within subjects on HD, the combination of increased CRP and reduced PON1 may identify subjects at a high risk for cardiovascular complications.

Plasmodium falciparum-infected erythrocytes and oxidized low-density lipoprotein bind to separate domains of CD36.

The cytoadherence of erythrocytes (red blood cells) infected with Plasmodium falciparum (pRBCs) to endothelial cells and the uptake of oxidized low-density lipoprotein (oxLDL) by macrophages are both mediated, in part, by the glycoprotein receptor CD36. The interaction of lipoproteins and pRBCs competing for the human CD36 receptor was examined by use of Chinese hamster ovary cells expressing human CD36. OxLDL competitively inhibits the adherence of pRBCs to CD36, but native LDL and high-density lipoprotein do not. Modification of Lys residues in CD36 inhibits both oxLDL and pRBC binding; however, only oxLDL binding is inhibited by receptor iodination, and only pRBC binding is influenced by pH variations and receptor reduction. Furthermore, peptide inhibitors of the pRBC/CD36 interaction do not influence oxLDL binding. These results suggest that, although oxLDL competitively inhibits the adherence of pRBCs, these ligands interact with distinct domains on the CD36 receptor.

Apolipoprotein E R112; R251G: a carboxy-terminal variant found in patients with hyperlipidemia and coronary heart disease.

A 49 year-old hypercholesterolemic male with marked electrocardiographic ST segment depression on exercise testing was found to have an apo E E3/3 phenotype by isoelectric focusing, but an APOE E4/3 genotype using HhaI restriction isotyping. DNA sequence analysis of the proband's APOE gene found a G-->C point mutation at codon 251. This predicted a change in the amino acid encoded by codon 251, from arginine to glycine. The mutation occurred on an allele that encoded arginine at position 112 and this variant was named APOE R112; R251G. The R251G change altered a recognition site for the endonuclease StuI and was the basis for a restriction isotyping method to rapidly screen for this mutation. In relatives of the proband, APOE R112; R251G was consistently found in subjects with both hyperlipidemia and atherosclerosis. Apo E R112; R251G-containing very low density lipoproteins bound normally to macrophages in vitro. However, the proband had an abnormal post-prandial lipoprotein response to a dietary fat challenge. The association of APOE R112; R251G with abnormal phenotypes suggests that the amino acid change in the carboxy-terminal, perhaps in combination with the common amino acid polymorphism at codon 112, has a functional impact upon lipoprotein metabolism in members of this family.

Identification of disulfide-linked apolipoprotein species in human lipoproteins.

We wished to determine whether apolipoprotein C-IIToronto, a mutant form of apolipoprotein C-II that contains a C-terminal cysteine residue, exists as a monomeric species or as multiple disulfide-linked species in plasma lipoproteins. The plasma lipoproteins from a heterozygous carrier and two homozygous carriers of apoC-IIToronto were investigated. The mutant apolipoprotein was found in homodimeric form and as heterodimers with apolipoprotein A-II, apolipoprotein B-100, and apolipoprotein E. Of particular interest was the demonstration of the existence of the disulfide-linked species apolipoprotein B-100:A-II and B-100:C-IIToronto in the very low density and low density lipoproteins in subjects who were carriers of apoC-IIToronto. We also observed that apoE3:C-IIToronto and apoE3:A-II dimers were present in the chylomicrons and very low density lipoproteins of these subjects. The observation of the existence of apolipoprotein B-100:A-II was extended to other hypercholesterolemic and hypertriglyceridemic subjects. The highest proportion of apolipoprotein B-100:A-II was observed in the very low density lipoproteins of hypertriglyceridemic subjects. The concentration of this species was significantly higher in hyperlipidemic subjects than in normolipidemic controls. These results demonstrate that the molecular species of cysteine-containing apolipoproteins are complex and should be considered in studies of human lipoprotein composition and function.

Beta-VLDL in hepatic lipase deficiency induces apoE-mediated cholesterol ester accumulation in macrophages.

Hepatic lipase-deficient subjects in the Ontario kindred are compound heterozygotes for hepatic lipase mutations (Ser267-->Phe and Thr383-->Met). Cholesteryl ester-rich beta-very-low-density lipoprotein (beta-VLDL) accumulates in plasma and such subjects have premature atherosclerosis. To determine a possible mechanism, we hypothesized that hepatic lipase-deficient beta-VLDL, homozygous for apolipoprotein (apo) E3, would cause cholesteryl ester accumulation and foam cell formation in macrophages. beta-VLDL and pre-beta-VLDL were isolated by Pevikon electrophoresis and incubated with J774 macrophages, cells that do not secrete apoE. beta-VLDL increased cellular cholesteryl ester content 13-fold, whereas pre-beta-VLDL increased cholesteryl ester sevenfold. beta-VLDL increased acyl CoA:cholesterol acyltransferase activity fourfold (measured as [14C]oleate incorporation into cholesteryl ester). Preincubation of hepatic lipase-deficient beta-VLDL with the anti-apoE monoclonal antibody 1D7, which inhibits binding of apoE to low-density lipoprotein receptors, inhibited cellular cholesteryl ester accumulation by 75%, whereas the anti-apoB blocking monoclonal antibody 5E11 failed to inhibit cellular cholesteryl ester accumulation. In contrast to hepatic lipase deficiency, beta-VLDL from type III subjects (E2/E2) failed to increase cellular cholesteryl ester or acyl CoA:cholesterol acyltransferase more than 1.5-fold. Thus, hepatic lipase-deficient beta-VLDL readily induces cholesteryl ester accumulation in J774 macrophages, a process mediated by functional apoE3. This may explain the premature atherosclerosis observed in this kindred.

Evidence that cholesteryl ester and triglyceride accumulation in J774 macrophages induced by very low density lipoprotein subfractions occurs by different mechanisms.

The present investigations have examined the mechanism(s) whereby Sf 60-400 very low density lipoproteins (VLDL) from Type IV hypertriglyceridemic subjects cause cholesteryl ester and triglyceride accumulation in J774 macrophages. Both apolipoprotein (apo) E-poor and apoE-rich Type IV VLDL subfractions, isolated by heparin-Sepharose chromatography, were capable of enhancing cellular cholesterol and triglyceride content. The apoE-rich fraction was significantly more effective at inducing cholesterol esterification (P < 0.05) and accumulation of esterified cholesterol (P < 0.05), whereas both subfractions caused similar increases in cellular triglyceride content. Thus, the amount of VLDL-associated apoE determined the extent to which Type IV VLDL loaded J774 cells with cholesterol but not triglyceride. Two VLDL subfractions, Sf 60-400 and Sf 20-60, isolated from Type III subjects homozygous for apoE2, caused little or no effect on cellular esterified cholesterol content, whereas both fractions induced the same degree of cellular triglyceride accumulation as Type IV VLDL. Type IV VLDL-induced cholesteryl ester accumulation was blocked by an anti-apoE monoclonal antibody, known to block the binding of apoE to the LDL receptor; however, the increase in cellular triglyceride was unaffected. Therefore, VLDL-induced triglyceride accumulation in this cell line can occur without apoE-mediated uptake of intact VLDL particles. The addition of heparin to J774 cells resulted in a 6-fold increase in lipoprotein lipase (LPL) activity in the media, and significantly enhanced the ability of Type IV VLDL to induce cellular triglyceride accumulation (P < 0.01), but significantly decreased cellular cholesteryl ester content (P < 0.025). Finally, Sf 60-400 VLDL from two subjects homozygous for apoC-II deficiency failed to increase cellular lipid content. However, the addition of exogenous apoC-II to C-II-deficient VLDL resulted in significant increases of both triglyceride and esterified cholesterol in J774 cells. In the presence of apoC-II, the anti-apoE monoclonal antibody blocked the cellular cholesteryl ester increase induced by C-II-deficient VLDL, but had no effect on the increase in cellular triglyceride. Collectively, these experiments demonstrate that extracellular lipolysis of Sf 60-400 VLDL by LPL is required for cholesteryl ester and triglyceride accumulation in J774 macrophages. After interaction with cellular LPL, VLDL triglycerides are hydrolyzed. The resulting free fatty acids are readily taken up by the macrophage, and re-esterified into triglyceride. Lipolysis proceeds until apoE epitopes are exposed, allowing the triglyceride-depleted remnant, containing all the cholesteryl ester, to be taken up via an apoE-mediated process.